The recent discovery and confirmation by four independent laboratories, including ours, that malignant gliomas are frequently associated with expression of human cytomegalovirus antigens provides a unique opportunity to harness the cytolytic power of the immune system to eradicate CMV-infected tumor cells without harming surrounding normal brain. Our efforts in enhancing CMV-specific immune responses using CMV pp65 RNA- pulsed dendritic cells (DCs) have demonstrated the capacity to enhance CMV-specific cellular and humoral responses and elicit promising radiographic and clinical responses in patients with newly-diagnosed GBM. Despite these encouraging results, we have found that patients with GBM elicit profound immunologic CMV- specific deficits at diagnosis that are characterized by the inability to mount effective polyfunctional T cell responses (simultaneous production of IL-2, IFN, TNF, and cytolytic granules (CD107)) upon stimulation with CMV antigens in vitro. Polyfunctional T cell responses have been shown to mediate the effective control of chronic viral infections such as HIV and CMV in humans, and while DC vaccination improved the frequency of monofunctional CMV-specific T cells, polyfunctional responses were not enhanced. Importantly, we have found that polyfunctional CMV-specific T cell responses could be restored in these same patients in vitro using pp65 RNA pulsed DCs coupled with addition of exogenous IL-2 or through the removal of CD4+CD25+FOXP3+ regulatory T cells (Tregs) prior to stimulation. The results suggest that reversal of cell-mediated deficits in vivo in patients with GBM may be a feasible goal and may significantly improve the efficacy of antitumor immunotherapy. In this proposal, we will explore methods to reverse cell-mediated deficits in patients with GBM using CMV RNA pulsed DCs co-transfected with RNAs encoding for cytokines that favorably modulate polyfunctional T cell responses and inhibit expansion of immunosuppressive Tregs. The use of RNA-modified DCs may allow for the targeted delivery of immunomodulatory cytokines without the induction of systemic toxicity or autoimmune risks of systemic Treg inhibition. These studies have significant potential to improve clinical outcomes for patients with GBM. PUBLIC HEALTH RELEVANCE: The significance of this research is that it may advance a new therapy for malignant brain tumors as well as provide a strategy for treatment that can be applied to many other cancers. Improved therapy for cancer has significant potential to improve public health and quality of life for patients affected by malignant disease.